The long-term goal of this research project is to understand the outer membrane (OM) secretion mechanism for autotransporter (AT) virulence proteins from Gram-negative bacterial pathogens. AT proteins represent the largest class of secreted virulence proteins from pathogens including Y. pestis, H. pylori, B. pertussis, enterohemorrhagic E. coli, Shigella, A. baumannii, K. pneumoniae, and N. meningitidis. Collectively, these pathogens kill >5 million people annually, and can be challenging to defeat using current antibiotics. Controlling the AT secretion mechanism would open up new avenues to control Gram-negative pathogens. But to gain such control, basic research is needed to define the specific mechanism used to deliver AT virulence proteins to the cell surface. In particular, it is unclear how these one-shot molecular machines are transported across the OM in the absence of ATP or a proton gradient. To best address this question, our proposed research combines biochemical, biophysical, genetic and structural methods to establish the energetic and kinetic principles underlying AT OM secretion. This research has three specific aims: (1) We will determine how the energy released upon folding of the mature AT virulence protein (the central passenger domain) affects OM secretion, specifically the interplay between AT passenger domain folding stability and kinetics, and C- versus N-terminal stability. (2) We will determine whether the AT passenger exits the cell through its own C-terminal porin domain, or through another OM porin such as BamA. (3) We will test the hypothesis that the energy landscape for AT passenger domain folding possesses features that disable initiation of folding from the N-terminus. Despite broad sequence and functional diversity, recent high- resolution structures have identified common structural features in AT passengers, and our own lab has identified common folding features. And, our laboratory has pioneered new biophysical assays to directly interrogate features of the AT OM secretion mechanism in vivo, establishing the current paradigm that OM secretion proceeds via C- to N-terminal transport of the passenger domain. Recently, we have determined that OM secretion efficiency can be controlled by regionalized stability within the passenger domain. These preliminary studies have established the conceptual framework and feasibility of these aims. Here, we will exploit our assay to quantitatively and reversibly arrest OM secretion intermediates to address previously unanswerable questions regarding the AT OM secretion mechanism. Together, these studies will identify the crucial features and most-easily-accessed attack points for this most common mechanism for virulence protein secretion. Due to the crucial role of AT proteins in bacterial virulence, the rapid rise of drug-resistant bacterial strains, and large number of fatalities due to Gram-negative bacterial infections, our proposed studies will have a large impact on molecular medicine and human health.